Botulism is a severe neuroparalytic disease caused by one of seven botulinum neurotoxins (BoNTs), produced by the anaerobic, spore-forming bacterium Clostridium botulinum. These protein neurotoxins are the most potent toxins known to man. There are BoNT toxoid vaccines available currently as Investigational New Drugs. However, due to the numerous shortcomings associated with the toxoid vaccines (i.e., dangerous to produce, high cost of manufacturing, high reactogenicity), there is an urgent need to develop new generation vaccines for the prevention of botulism. The goals of this research are to develop a new botulism vaccine using the carboxyl-terminal 50 kDa C-fragments (Hc) of the heavy chains in BoNTs as antigens and to study the delivery of the vaccine utilizing a replication-defective adenoviral vector via the intranasal and transcutaneous routes. These non-invasive vaccine delivery methods will undoubtedly enhance the compliance of a vaccination program, which is especially critical in response to a potential bioterrorist attack using BoNTs. After construction of replication-defective adenoviral vectors encoding the immunogenic C-fragments of the heavy chains in BoNTs, vaccination protocols in mice comparing the intranasal and transcutaneous delivery modes with the subcutaneous injection of the currently available pentavalent botulinum toxoid vaccine (PBT) will be studied. The specific aims of this project are: Specific Aim #1: To construct replication-defective adenoviral vectors encoding the C-fragments of the heavy chains in BoNTs. Specific Aim #2: To study the mucosal and systemic immunity elicited by the vectored vaccine developed in aim #1 through intranasal and transcutaneous immunization in a mouse model.